US8688427B2 - Enzyme catalysts for diels-alder reactions - Google Patents

Enzyme catalysts for diels-alder reactions Download PDF

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US8688427B2
US8688427B2 US13/130,260 US200913130260A US8688427B2 US 8688427 B2 US8688427 B2 US 8688427B2 US 200913130260 A US200913130260 A US 200913130260A US 8688427 B2 US8688427 B2 US 8688427B2
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David Baker
Alexandre Zangellini
Justin Siegel
Jennifer St. Clair
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/08—Phosphoric triester hydrolases (3.1.8)
    • C12Y301/08002—Diisopropyl-fluorophosphatase (3.1.8.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/03—Intramolecular oxidoreductases (5.3) transposing C=C bonds (5.3.3)
    • C12Y503/03001—Steroid DELTA-isomerase (5.3.3.1)

Definitions

  • the Diels-Alder reaction is a cycloaddition reaction between a conjugated diene and a substituted alkene or alkyne (e.g., a dienophile), to form a substituted cyclohexene system.
  • the reaction forms two carbon-carbon bonds and up to four new stereogenic centers in one step. Since its discovery, the Diels-Alder reaction has been a cornerstone reaction for the synthesis of organic compounds.
  • RNA catalysts Two different families of synthetic ribozymes (RNA catalysts) have been engineered to date.
  • a library of RNA molecules was created covalently attached to an acyclic diene and selected for Diels-Alder activity (41,42).
  • the best ribozymes showed rate enhancements of up to 800-fold over the uncatalyzed reaction. Since one of the substrates is covalently attached to the RNA catalyst, the reaction is effectively first order and the author reported a k cat /K M of 3.95 M ⁇ 1 s ⁇ 1 and an effective molarity of 2M. Similar results were obtained using a library of PEG-ylated RNA molecules attached to anthracene and directed to catalyze a Diels-Alder reaction with a maleimide dienophile (43).
  • antibody 39-A11 catalyzes the Diels-Alder reaction between an electron-rich acyclic diene and an N-aryl maleimide (46), although its proficiency is lower due to a less complimentary binding pocket.
  • antibodies 4D5, 13G5, and several others were shown to catalyze regio-, diastereo- and enantio-selective addition of 4-carboxybenzyl trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide, a model Diels-Alder reaction (10).
  • the present invention provides enzyme catalysts for Diels-Alder reactions, including intermolecular Diels-Alder reactions, as well as protein scaffolds for making such enzyme catalysts.
  • the invention provides methods of making the enzyme catalysts, including by de novo computational design. The present invention thereby provides enzyme catalysts capable of catalyzing a desired Diels-Alder reaction, including with a specified or desired stereo-selectivity.
  • the present invention provides enzyme catalysts for Diels-Alder reactions.
  • the enzymes have scaffolds derived from non-immunoglobulin amino acid sequences, such as diisopropylfluorophosphatase from Loligo vulgaris (PDB-ID 1E1A) (or a homolog thereof), or ketosteroid isomerase Pseudomonas putida (PDB-ID 1OHO) (or a homolog thereof).
  • Alternative exemplary scaffolds are disclosed herein in Table 1.
  • the Diels-Alder scaffold has a pocket designed and/or adapted to catalyze a Diels-Alder reaction, as disclosed in detail herein.
  • the enzyme active site comprises amino acid side chains positioned to act as electron-withdrawing and/or electron-donating groups to stabilize the Diels-Alder transition state, and/or contains a substrate-binding pocket having hydrophobic and/or polar interfaces that accommodate the Diels-Alder reaction by binding the substrates in the proper orientation for catalysis.
  • the active site comprises one or more amino acid side chains positioned to stabilize the Diels-Alder transition state by hydrogen-bond acceptor and/or donor groups.
  • the active site may contain an amino acid residue having a side chain that stabilizes the Diels-Alder transition state by accepting a hydrogen-bond from the diene portion of the transition state, or from the dienophile portion of the transition state.
  • such an amino acid residue may be at the position corresponding to position 195 of the diisopropylfluorophosphatase scaffold (SEQ ID NO:2).
  • An amino acid residue having a side chain that stabilizes the Diels-Alder transition state by donating a hydrogen-bond to the dienophile portion of the transition state, or to the diene portion of the transition state, may be at the position corresponding to position 121 of the scaffold of SEQ ID NO:2.
  • the active site accommodates the Diels-Alder substrates in the proper orientation for catalysis.
  • the active site may contain an amino acid residue having a side chain that accepts a hydrogen bond from the Diels-Alder transition state, and such amino acid may be at the position corresponding to position 82 of the scaffold (e.g., SEQ ID NO:9).
  • the active site may also contain an amino acid residue that donates a hydrogen-bond to the Diels-Alder transition state, and such amino acid residue may be at the position corresponding to position 114 of the scaffold (e.g., SEQ ID NO:9).
  • the active site accommodates the Diels-Alder substrates in the proper orientation for catalysis.
  • the Diels-Alder enzyme catalyst in addition to having the catalytic amino acid(s) on the protein scaffold in the proper position(s) to stabilize the Diels-Alder transition state, also contains an active site pocket with a tight complementary surface to sufficiently accommodate, not only the desired substrate(s), but also the transition state and resulting product.
  • the active site may provide additional contacts to the substrate(s), and/or provide a complementary interface with the proper positioning of polar and non-polar amino acid side chains.
  • the active site pocket is a relatively non-polar environment (e.g., hydrophobic), lined predominately with non-polar amino acids.
  • amino acid residues that may influence the position of the catalytic side chains and/or influence the environment and/or shape of the active site include amino acid residues corresponding to positions 21, 36, 37, 39, 72, 74, 90, 120, 135, 136, 144, 146, 148, 149, 173, 175, 176, 196, 225, 229, 230, 244, 269, 271, 272 and 287 of the scaffold (SEQ ID NO:2).
  • amino acid residues that may influence the position of the catalytic side chains and/or influence the environment and/or shape of the active site include amino acid residues corresponding to positions 86, 93, 95, 121, 118, 116, 40, 43, 39, 84, 62, 59, 58, 37, 46, 55, 97, 114, 112, 99, 82, 19, 60, 64, 16, 31, 15, 80, 27, 56.
  • the invention provides methods for making enzymes that catalyze Diels-Alder reactions.
  • the methods may involve de novo enzyme design by transition state, substrate and/or product model placement within a scaffold or scaffold library, or in other embodiments, the methods involve designing the scaffolds and Diels-Alder enzymes described herein (or homologs thereof) for desired substrates.
  • the method comprises first creating a Diels-Alder transition state, substrate, and/or product model with protein functional groups positioned to support catalysis.
  • a protein scaffold is then identified (e.g., using hashing algorithms) that has backbone coordinates sufficient to support the positioning of the protein functional groups. Mutations are then introduced into the scaffold by rational design and/or as guided by functional Diels-Alder assays to create and improve the designs.
  • the invention involves making Diels-Alder enzymes by mutation of the scaffolds and enzymes described herein (or homologs thereof) to accommodate desired Diels-Alder substrates.
  • FIG. 1 illustrates the basic computational design methodology, including transition state design with catalytic functional groups, scaffold identification, followed by further design, and then optimization of the enzyme by targeted mutation and guided by functional assay.
  • FIG. 2 illustrates a Diels-Alder reaction. Diene (1) and dienophile (2) undergo a pericyclic [4+2] cycloaddition (3) to form a chiral cyclohexene ring (4). Schematic of the design target active site, with hydrogen bond acceptor and donor groups activating the diene and dienophile and a hydrophobic pocket surrounding the reaction core, is depicted in (3).
  • FIG. 3 shows the dihedrals in the diene molecule which were allowed to vary in order to generate the conformer ensembles during computational design.
  • FIG. 4A shows the first minimal active site used for design.
  • the two catalytic residues are an Asparagine/Glutamine as a hydrogen bond acceptor on the carbamate nitrogen, and a Serine as a hydrogen bond donor to the diene carbonyl.
  • the final minimal active site contains a consensus between the substrate, transition state and product structures, but only a symbolic representation of the transition state structure is represented here.
  • FIG. 4B shows the second minimal active site used for design.
  • the two catalytic residues are an Asparagine/Glutamine as a hydrogen bond acceptor on the carbamate nitrogen, and a Tyrosine as a hydrogen bond donor to the diene carbonyl.
  • the final minimal active site contains a consensus between the substrate, transition state and product structures, but only a symbolic representation of the transition state structure is represented here.
  • FIG. 5 shows the geometrical parameters used to place the first catalytic side-chain functional group in active site descriptions.
  • the idealized geometries are listed, as well as the deviation (tolerance) used both for generating the discrete ensemble of rigid-body placements of transition state consensus structures, and for defining the constraint energy for minimization.
  • FIG. 6 shows the geometrical parameters used to place the second catalytic side-chain functional group in active site descriptions.
  • the idealized geometries are listed, as well as the deviation (tolerance) used both for generating the discrete ensemble of rigid-body placements of transition state consensus structures, and for defining the constraint energy for minimization.
  • FIG. 7 shows the sequence of the scaffold aligned with the original design for the Diels-Alder catalyzing enzyme (DA — 20 — 00, SEQ ID NO:4)).
  • the scaffold is diisopropylfluorophosphatase from Loligo vulgaris (PDB-ID 1E1A).
  • the amino acid sequence for the native scaffold is provided herein as SEQ ID NO:2.
  • the catalytic residues in the original design are shaded, and amino acids mutated during the initial design stages are shown in bold.
  • FIG. 8 illustrates the general structure of the scaffold.
  • Diisopropylfluorophosphatase from Loligo vulgaris may generally be described as a 6-bladed beta barrel propeller scaffold (PDB-ID 1E1A).
  • FIG. 9 shows the amino acid sequence of the scaffold with regional secondary structures.
  • the scaffold contains about 51% beta-sheet (shown by arrows). Turns are also illustrated. Triangles indicate the location of a beta-bridge. A three-amino acid helix is located near the C-terminus.
  • FIG. 10 illustrates the structure of the computationally designed Diels-Alderase.
  • Top panel (A) surface view of the design model (DA — 20 — 00) bound to the substrates (diene and dienophile). The catalytic residues making the designed hydrogen bonds are depicted as sticks.
  • Middle panel (B) overlay of the design model (DA — 20 — 00) and the apo enzyme crystal structure of DA — 20 — 00_A74I.
  • FIG. 11 shows the combinatorial mutants from round 1 active site optimization.
  • the observed velocity for each mutant was normalized by the velocity of DA — 20 — 00 in order to determine the effective rate enhancement conferred by each mutation.
  • the reactions were done at 1 mM diene, and 10 mM dienophile, PBS, 298K, and 100 ⁇ M protein.
  • FIG. 12 shows an alignment between DA — 20 — 00, the initial design, and DA — 20 — 04, the enzyme after one round of optimization.
  • the catalytic residues Y121 and Q195 are shown shaded.
  • Three mutations were made relative to the initial enzyme: A21I and A74I which are thought to improve packing around the transition state, and S271A, which is thought to make the dienophile environment more non-polar. The three mutations are shown in bold type.
  • FIG. 13 shows the relative activity of selected second round mutants. Mutations showing enhanced activity on DA — 20 — 04 were sequenced, grown large scale, and assayed. The assay was done at 0.1 mM dieine, 3 mM dienophile, PBS, 298K, and 20 ⁇ M protein.
  • FIG. 14 shows an alignment between DA — 20 — 04 and DA — 20 — 10, the enzyme after the second round of optimization.
  • the catalytic residues Y121 and Q195 are shown shaded.
  • Three mutations were made relative to the DA — 20 — 04 enzyme: A173C, which is thought to improve packing around the catalytic glutamine; Q149R, which is thought to hydrogen bond to the carboxylate on the diene; and A272N, which is thought to hold the catalytic tyrosine in proper conformation for catalysis.
  • the three mutations are shown in bold type.
  • FIG. 15 shows kinetic characterization of DA — 20 — 10.
  • A Dependence of reaction velocity on diene concentration for different fixed dienophile concentrations. The diene concentration was varied from 3.0 to 0.18 mM with a fixed concentration of 100 mM ( ⁇ ), 66 mM ( ⁇ ), 44 mM ( ⁇ ), 30 mM ( ⁇ ), 20 mM ( ⁇ ), or 13 mM ( ⁇ ) dienophile
  • B Dependence of reaction velocity on dienophile concentration for different fixed diene concentrations.
  • the dienophile was varied from 100 to 13.0 mM with a fixed concentration of 3 mM ( ⁇ ), 2 mM ( ⁇ ), 1.3 mM ( ⁇ ), 0.9 mM ( ⁇ ), 0.6 mM ( ⁇ ), 0.4 mM ( ⁇ ), 0.26 mM ( ⁇ ), or 0.18 mM ( ⁇ ) dienophile. Reactions were carried out with 20 ⁇ M protein in PBS at 298 K.
  • FIG. 16 shows DA — 20 — 10 Turnover Number.
  • the reaction was carried out in 3 mM diene, 100 mM dienophile, 25 ⁇ M enzyme, in PBS at 298K. Turnover number was calculated by subtracting the product formed in the background reaction from the product formed in the enzymatic reaction. This enzyme-produced product was then divided by the protein concentration to determine turnover number.
  • FIG. 17 shows production with DA — 20 — 10 and effects of catalytic residues.
  • the reaction was carried out in 1 mM diene, 50 mM dienophile, 200 ⁇ M enzyme, in PBS at 298K.
  • the proteins assayed are DA — 20 — 10 ( ⁇ ), Y121F ( ⁇ ), Q195E (x), and tela scaffold (*), uncatalyzed ( ⁇ ).
  • FIG. 18 shows the absolute stereoselectivity of DA — 20 — 10.
  • the transition states which lead to the four possible ortho-stereoisomers are shown above the reaction chromatograms.
  • Background reaction 2 mM diene and 70 mM dieneophile in a PBS solution for 24 hours at 298 K.
  • DA — 20 — 10 reaction 50 ⁇ M protein, 0.5 mM diene, and 10 mM dieneophile in a PBS solution for 48 hours at 298K.
  • the four stereoisomers were separated using liquid chromatography (11), and samples injected onto a Daicel AD-H column.
  • FIG. 19 illustrates the substrate specificity of DA — 20 — 10 and a single mutant of DA — 20 — 10, H287N. Reactions were carried out with 0.2 mM diene 1 ( FIG. 1 ), and 10 mM of one of the six dienophiles depicted in PBS at 298K in the absence or presence of 60 ⁇ M DA — 20 — 10 or DA — 20 — 10_H287N. The depicted values in the figure are the mean (bars) and standard deviation (error bars) of four independent measurements of the product peak area (arbitrary units) formed per hour, determined using a liquid chromatography-mass spectroscopy assay.
  • DA — 20 — 10_H287N is most active on dienophile 2E, which is suboptimal for DA — 20 — 10.
  • FIG. 20 shows the amino acid sequence (SEQ ID NO:9) for a second Diels-Alder enzyme catalyst, prepared from a ketosteroid isomerase scaffold. This enzyme also catalyzes the reaction illustrated in FIG. 2 . Active site residues are shown in bold, and catalytic residues are indicated (in blue).
  • FIG. 21 illustrates exemplary reactions catalyzed by a Diels-Alder enzyme catalyst.
  • Reaction (1) produces an intermediate used for the production of TAMIFLU.
  • Reaction (2) leads to the production of a building block useful for the manufacture of the polymer PET.
  • Reaction (3) can be used for the manufacture of vitamin B6.
  • Reaction (4) is a reaction commonly referred to as “click chemistry.”
  • the Diels-Alder reaction generally takes place between a conjugated diene and a dienophile to produce a 1,4-addition product (with respect to the diene).
  • the Diels-Alder reaction is a particular example of 4+2 sigmatropic cycloadditions.
  • the term “dienophile” refers to a group or bond that is attracted to the diene.
  • the reaction mechanism is a single step involving a cyclic redistribution of bonding electrons.
  • the reaction may be favored by the presence of electron-withdrawing groups on the dienophile, and by electron-donating groups on the diene.
  • Frontier Orbital Theory shows that interaction of the highest occupied molecular orbital (HOMO) of the diene with the lowest unoccupied molecular orbital (LUMO) of the dienophile stabilizes the transition state and leads to charge transfer from diene to dienophile (30).
  • Frontier Orbital Theory also shows that a Diels-Alder reaction rate can be increased with the interaction of the lowest unoccupied molecular orbital (LUMO) of the diene with the highest occupied molecular orbital (HOMO) of the dienophile, leading to what is known as an inverse electron demand Diels-Alder reaction.
  • the interaction between the HOMO and LUMO, and the stability of the transition state relative to the ground state, may be increased by an electron donating effect to the diene (which increases the HOMO energy and stabilizes the positive charge accumulating in the transition state), and by an electron-withdrawing effect to the dienophile (which lowers the LUMO energy and stabilizes the negative charge accumulating in the transition state), or alternatively the converse for inverse electron demand reactions.
  • any functional group from the protein side that will create or increase electron-withdrawing and electron-donating effects is predicted to increase the rate of the Diels-Alder reaction.
  • intramolecular and intermolecular Diels-Alder reaction rates may be enhanced by proximity effects, such as binding of the substrate or substrates into an enzyme active site so that the effective concentration of the substrate or substrates is higher than in water.
  • a hydrophobic binding pocket that binds the core of the two substrates in the optimal relative orientation with appropriately placed hydrogen bond donors and acceptors is believed to be an effective Diels-Alder catalyst, because of both orbital and proximity effects.
  • the present invention provides enzyme catalysts for Diels-Alder reactions.
  • the enzymes generally have scaffolds derived from non-immunoglobulin amino acid sequences.
  • the scaffolds have active site “pockets” of sufficient size and shape to accommodate the desired Diels-Alder substrate(s).
  • Exemplary scaffolds include diisopropylfluorophosphatase from Loligo vulgaris (and homologs thereof), which may generally be described as a 6-bladed beta barrel propeller scaffold (PDB-ID 1E1A), and ketosteroid isomerase from Pseudomonas putida (and homologs thereof), which may generally be described as a alpha+beta roll (PDB-ID 1OHO).
  • the structure of the native scaffold is depicted in FIG. 8 .
  • the amino acid sequence for the native scaffold is provided herein as SEQ ID NO:2 (see FIGS. 7 and 9 ).
  • the Diels-Alder catalysts of the invention are based upon this scaffold, with a Diels-Alder active site grafted onto the scaffold by mutation (e.g., substitution, deletion, and/or insertion of amino acid residues) as described in detail herein.
  • the active site generally comprises amino acid side chains to act as electron-withdrawing and/or electron-donating groups to the Diels-Alder transition state.
  • the electron-withdrawing and electron-donating groups may be hydrogen bond donor and/or hydrogen bond acceptor groups in the proper orientation to stabilize a Diels-Alder transition state.
  • the Diels-Alder catalyst contains a substrate-binding pocket having a complementary interface of non-polar and/or polar amino acid residues to accommodate the Diels-Alder reaction. See FIG. 2 .
  • the Diels-Alder catalyst may alternatively be based upon a homolog of the scaffold, including homologs of the scaffolds described in Table 1, and homologs of SEQ ID NO:2 and SEQ ID NO:9.
  • sequence homologs may be defined by a significant level of sequence identity or similarity between two sequences (e.g., between a particular scaffold and potential homolog). The level of identity or similarity can be determined using any suitable alignment tool known in the art, including Tatusova et al., Blast 2 sequences—a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 174:247-250 (1999).
  • the homolog is a structural homolog, which may or may not exhibit detectable sequence identity or similarity with the scaffold of interest.
  • structural homologs are protein sequences that share a very similar 3-dimensional tertiary structure.
  • Structural homologs may be detected from structural databases by algorithms known as structural alignments. Examples of such programs that can be used to detect structural homologs include DALI/Dalillte (L. Holm and C. Sander (1996) Science 273(5275):595-60); Mammoth/Maxsub (D. Lupyan et al. (2005) A new progressive - iterative algorithm for multiple structure alignment, Bioinformatics ), as well structurally annotated database such as SCOP (Andreeva et al.
  • the scaffold homologs or catalysts of the invention will have a level of identity to the scaffolds described herein of at least 20%, 30%, 40%, 50%, 60%, 70% 80%, 90%, 95%, or 98% (e.g., to SEQ ID NO:2, SEQ ID NO:9, or an enzyme disclosed in Table 1). With this level of homology, the enzyme maintains the basic tertiary fold of the scaffold and has Diels-Alder activity for a desired substrate (including substrates described herein), by mutation of active site residues.
  • the scaffold homolog or catalyst is a structural homolog of a scaffold described herein, that is, the scaffold homolog or catalyst shares a similar 3-dimensional tertiary structure to the scaffolds described herein, as may be determined using the structural alignment tools described above.
  • the Diels-Alder catalyst has an active site comprising amino acid side chains positioned to stabilize a Diels-Alder transition state by electron-withdrawing effects of one or several hydrogen-bond donor(s) (interacting with the dienophile, or the diene in the case of inverse electron demand Diels-Alder reactions), and electron-donating effects of one or several hydrogen-bond acceptor(s) (interacting with the diene or dienophile).
  • the amino acid having a side chain accepting a hydrogen-bond from the transition state may be at the position corresponding to position 195 of SEQ ID NO:2.
  • the amino acid having a side chain donating a hydrogen-bond to the transition state may be at the position corresponding to position 121 of SEQ ID NO:2.
  • any amino acid residue lining the active site may carry functional side chains to provide electron withdrawing or electron donating effects to the Diels-Alder transition state.
  • Such amino acids may be identified for a particular scaffold and with respect to a particular Diels-Alder transition state, using computational tools known in the art and described herein.
  • the amino acid at the position corresponding to position 195 of SEQ ID NO:2 has a side chain that is capable of stabilizing a Diels Alder transition state.
  • the amino acid residue at the position corresponding to position 195 of SEQ ID NO:2 may be any amino acid having a side chain capable of accepting a hydrogen bond from the diene, such as from a substituent of the diene containing a hydroxyl or NH group (e.g., carbamate NH). Exemplary diene substituents are described herein.
  • the amino acid at the position corresponding to position 195 of SEQ ID NO:2 is asparagine or glutamine.
  • the amino acid residue at the position corresponding to position 195 of SEQ ID NO:2 may be glutamine.
  • the amino acid at the position corresponding to position 121 of SEQ ID NO:2 has a side chain capable of stabilizing a Diels-Alder transition state.
  • the amino acid residue at the position corresponding to position 121 of SEQ ID NO:2 may act as a hydrogen bond donor to an electron-withdrawing group of the dienophile.
  • the amino acid corresponding to position 121 of SEQ ID NO:2 may hydrogen bond with a substituent of the dienophile having the formula —C(O)R, where R is H or a substituent as described in greater detail herein.
  • the amino acid at the position corresponding to position 121 of SEQ ID NO:2 is tyrosine, threonine, or serine.
  • the amino acid residue at the position corresponding to position 121 of SEQ ID NO:2 may be tyrosine.
  • amino acid residues lining the pocket are designed to achieve hydrophobic packing, and hence rate enhancement through proximity effects of substrate binding, and without electron withdrawing/donating effects.
  • halogen substituents e.g., Cl, Br, I
  • hydrophobic packing around the transition state is predicted to provide rate enhancement through proximity effects.
  • the Diels-Alder enzyme catalyst in addition to having the catalytic amino acid(s) on the protein scaffold in the proper position(s) to stabilize/bind the Diels-Alder transition state, also contains an active site pocket with a tight complementary surface to sufficiently accommodate, not only the desired substrate(s), but also the transition state and resulting product.
  • the active site may provide additional contacts to the substrate(s), and/or provide a complementary interface with the proper positioning of polar and non-polar amino acid side chains.
  • the active site may be designed using any publicly available software, such as ROSSETTADESIGN.
  • the active site pocket is a relatively non-polar environment (e.g., hydrophobic), lined substantially with non-polar amino acids.
  • amino acid residues lining the pocket may be defined as amino acid residues whose C ⁇ is within 8 ⁇ of any atoms of the transition state structure, or whose C ⁇ is within 10 ⁇ of any such atoms but with a C ⁇ -C ⁇ vector pointing toward the transition state structure.
  • residues in the Diels-Alder catalyst are any hydrophobic or polar residue, excluding Gly and Pro (e.g., AILVYWFCMHQNST). Charged residues are allowed in positions in the scaffold where the same charge residue is present in the wild-type (or starting) structure.
  • the pocket is lined with from about 3 to about 15 alanine residues, or from about 5 to about 10 alanine residues.
  • Amino acid residues that may carry catalytic side chains, may influence the position of the catalytic side chains, and/or influence the environment and/or shape of the active site include amino acid residues corresponding to positions 21, 36, 37, 39, 72, 74, 90, 120, 135, 136, 144, 146, 148, 149, 173, 175, 176, 196, 225, 229, 230, 244, 269, 271, 272 and 287 of SEQ ID NO:2.
  • amino acid residues that may carry catalytic side chains, or which may influence the position of the catalytic side chains, and/or which may influence the environment and/or shape of the active site include amino acid residues corresponding to positions 86, 93, 95, 121, 118, 116, 40, 43, 39, 84, 62, 59, 58, 37, 46, 55, 97, 114, 112, 99, 82, 19, 60, 64, 16, 31, 15, 80, 27, 56. of SEQ ID NO:9.
  • the Diels-Alder catalyzing enzyme may contain other amino acid substitutions that may be desirable for a particular Diels-Alder substrate or set of substrates, or which do not disrupt the shape of the active site or position of catalytic substrates.
  • additional amino acid substitutions do not significantly disrupt local secondary structures, as shown diagrammatically in FIG. 9 for the scaffold of SEQ ID NO:2.
  • the effect of amino acid substitution, insertion, and/or deletion on the enzyme structure can be reasonably predicted with available computational tools, including ROSSETTADESIGN.
  • the coordinates for the scaffolds are publicly available.
  • the Diels-Alder catalyst has from about 5 to about 30 amino acid substitutions with respect to the scaffold (SEQ ID NO:2), and is capable of catalyzing a Diels-Alder reaction.
  • the Diels-Alder catalyst has from about 10 to about 25, or from about 10 to about 20 amino acid substitutions with respect to SEQ ID NO:2.
  • the Diels-Alder enzyme catalyst may also contain one or more amino acid insertions or deletions (e.g., collectively from about 1 to 30, 1 to 20, 1 to 10, or from about 1 to 5, e.g., 1, 2, or 3) with respect to the scaffold, so long as these insertions or deletions do not affect the overall integrity of the active site.
  • such insertions or deletions may be positioned at or near the N- and/or C-termini, to create truncated scaffolds and/or enzymes comprising the active site.
  • the Diels-Alder catalyst has the amino acid sequence of SEQ ID NO:4 (DA — 20 — 00), or optionally with from 1 to about 10 amino acid substitutions, insertions, and/or deletions (collectively) with respect to SEQ ID NO:4.
  • the Diels-Alder catalyst in accordance with these embodiments contains the catalytic residues at positions corresponding to positions 121 and 195 of SEQ ID NO:4, and otherwise has been modified in the substrate bonding pocket, to bind the Diels-Alder substrates of choice.
  • the Diels-Alder catalyst has the amino acid sequence of SEQ ID NO:6 (DA — 20 — 04), or optionally with from 1 to about 10 amino acid substitutions, insertions, and/or deletions (collectively) with respect to SEQ ID NO:6.
  • the Diels-Alder catalyst in accordance with these embodiments contains the catalytic residues at positions corresponding to positions 121 and 195 of SEQ ID NO:6, and otherwise has been modified in the substrate bonding pocket to bind the Diels-Alder substrates of choice.
  • the Diels-Alder catalyst has the amino acid sequence of SEQ ID NO:8 (DA — 20 — 10), or optionally with from 1 to about 10 amino acid substitutions, insertions, and/or deletions (collectively) with respect to SEQ ID NO:8.
  • the Diels-Alder catalyst in accordance with these embodiments contains the catalytic residues at positions corresponding to positions 121 and 195 of SEQ ID NO:8, and otherwise has been modified in the substrate bonding pocket to bind the Diels-Alder substrates of choice.
  • the Diels-Alder catalyst has the amino acid sequence of SEQ ID NO:9, or optionally with from 1 to about 20, or 1 to about 10 amino acid substitutions, insertions, and/or deletions (collectively) with respect to SEQ ID NO:9.
  • the Diels-Alder catalyst in accordance with these embodiments may be modified in the substrate binding pocket to bind the Diels-Alder substrates of choice.
  • the Diels-Alder catalyst may be designed to stabilize the transition state for a variety of Diels-Alder reactants. That is, amino acid substitutions, insertions and/or deletions with respect to the scaffold, or with respect to the Diels-Alder catalyzing enzymes of SEQ ID NOS: 4, 6, 8, or 9 may be selected with regard to a particular Diels-Alder reaction.
  • the enzyme catalyzes a reaction with a linear or cyclic diene.
  • the diene may be a five- or six-membered ring having two conjugated double bonds, and the ring may have multiple heteroatoms (typically 1 or 2), such as N, O, and/or S.
  • heteroatoms typically 1 or 2
  • the diene must not be covalently locked into the trans conformation, so as to support the Diels-Alder reaction.
  • the diene may be substituted, for example, with up to four substituents.
  • the identity of the substituents will depend on the desired product, as well as the anticipated catalytic mechanism.
  • Exemplary diene substituents have less than about 30, about 20, or about 15 atoms, and may contain one or more (e.g., 1-10) heteroatoms selected from O, N, and/or S.
  • Exemplary dienes may have substituents independently selected from halogen (e.g., independently selected from Cl, Br, Fl, I), haloalkyl, alkyl (e.g., C1-C5), —C(O)O, —C(O)OR, —COR, —CN, —C(R)N, —CH(O), —C(O)—, aryl (e.g., substituted or unsubstituted phenyl), OH and —OR.
  • R may be any group, for example, containing about 20 or fewer atoms (e.g., 10 or fewer atoms).
  • R may represent a hydrogen or a carbon-containing substituent such as C1-5 alkyl (e.g., methyl), and may optionally include one or more (e.g., 1-3) heteroatoms independently selected from O, N, or S.
  • C1-5 alkyl e.g., methyl
  • heteroatoms independently selected from O, N, or S.
  • At least one substituent is an electron-donating group, which favors the Diels-Alder reaction, and which may interact with the Diels-Alder enzyme catalyst to stabilize the Diels-Alder transition state.
  • the diene may have a substituent defined by —N(H)R, or a substituent allowing for a similar hydrogen-bonding geometry with the hydrogen bond acceptor as exemplified by the position corresponding to position 195 of SEQ ID NO:2 (e.g., Q195).
  • R may be as defined above, or as described elsewhere herein.
  • at least one substituent is an electron-withdrawing group.
  • the dienophile may also be substituted, and the identity of such substituents will depend on the desired product as well as the intended catalytic mechanism.
  • the dienophile in various embodiments is substituted with one or more electron-withdrawing groups, making the dienophile more electrophilic.
  • the dienophile may be linear or cyclic, and generally includes an alkene or alkyne.
  • the substituent may be any group having less than about 30, about 20, or about 15 atoms, and may be suitable for withdrawing electron density from the carbon-carbon double or triple bond.
  • the electron-withdrawing group may contain one or more (e.g., 1-5) heteroatoms including O, N, and/or S.
  • the dienophile may be substituted with one or more electron-donating groups.
  • An electron-donating group may contain one or more (e.g., 1-5) heteroatoms including O, N, and/or S.
  • the dienophile has one or more substituents independently selected from halogen (e.g., independently selected from Cl, Br, Fl, I), haloalkyl, —C(O)OR, —COR, —CN, —C(R)N, —CH(O), —C(O)—, aryl (e.g., substituted or unsubstituted phenyl), OH and OR.
  • R may be any group, for example, containing about 20 or fewer atoms (e.g., 10 or fewer) and supporting the electron-withdrawing capacity of the overall substituent.
  • R may represent a hydrogen or a carbon-containing substituent such as C1-5 alkyl (e.g., methyl), and may optionally include one or more (e.g., 1-3) heteroatoms independently selected from O, N, or S.
  • C1-5 alkyl e.g., methyl
  • heteroatoms independently selected from O, N, or S.
  • the dienophile has a substituent defined by —C(O)R, where R is H, linear or branched alkyl (e.g., C1-C5), or N(R) 2 .
  • the dienophile may have a substituent allowing for a similar hydrogen-bonding geometry with a hydrogen-bond donor at position 121 (e.g., Y121) of the Diels-Alder enzyme catalyst.
  • the dienophile is a substituted or unsubstituted C1-4 alkene, such as ethene or ethyne, 1,2 di-chloroethene or 2-buten-1,4-ol as shown in FIG. 21 .
  • the dienophile may be a di-substituted alkene or alkyne, and may be in either cis or trans configuration. The stereochemistry of the dienophile will be maintained in the resulting product.
  • the Diels-Alder catalyst is designed to provide the desired stereochemistry.
  • the substrates providing the desired stereochemistry for the resulting Diels-Alder product are first selected for active site design or optimization. While there can be regio-, diastereo-, but not enantioselectivity in the uncatalyzed reaction, the present application shows that the Diels-Alder catalyzing enzyme is regio, diastereo, and enantioselective, and that stereoselectivity can be controlled by the enzyme.
  • the exemplified designs are based on one diastereo and one enantiomer of the Diels-Alder reaction between 4-carboxybenzyl trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide; namely, an endo 3R4S isomer.
  • Enzymes may be designed to catalyze products with alternative stereochemistry.
  • the Diels-Alder catalyzing enzyme may produce various products, such as products illustrated in FIG. 21 , with or without introduction of chiral centers.
  • reaction (1) produces an intermediate used for the synthesis of TAMIFLU, where the active is one of 32 possible stereoisomers.
  • the Diels-Alder enzyme may provide tight hydrophobic binding of 1,2-dichloro ethene, together with H-bonding of the hydroxyl group on C4 of the diene, and charged H-bonding with the carboxylate moiety on the C2 of the diene.
  • Reaction (2) is a Diels-Alder reaction that leads to one of the building blocks used for the manufacture of the polymer PET.
  • reaction (3) is a Diels-Alder reaction to manufacture vitamin B6. No enzyme exists in nature that catalyzes such a Diels-Alder reaction.
  • the current synthetic industrial route uses high temperature (around 200° C.) and inorganic catalysts.
  • the diene may be positioned, and the transition state stabilized, by making hydrogen bonds to the carboxylate and hydroxyl substituents.
  • Reaction (4) represents what is commonly known as “click chemistry”. With R being variable, this bimolecular reaction can be used to generate libraries of compounds, e.g., for combinatorial chemistry. The reaction introduces chiral centers and thus it would be extremely beneficial to control the stereoselectivity with a custom-designed enzyme. Furthermore, the usage of custom-designed enzymes would remove the need to use copper catalysts. A strategy similar to that for reactions 2 and 3 may be used to stabilize the transition state for reaction 4 depending on the substituent R.
  • the invention provides methods for making enzymes that catalyze Diels-Alder reactions.
  • the methods may involve de novo enzyme design by transition state, substrate or product placement within a scaffold or scaffold library, or in other embodiments, the methods involve designing the scaffolds and Diels-Alder enzymes described herein (SEQ ID NOS:2, 4, 6, 8, 9), and homologs thereof, for desired substrates.
  • the method comprises first identifying functional reactive sites (e.g., substrate binding residues) required to promote the desired Diels-Alder reaction; followed by the use of hashing algorithms to identify potential protein backbone structures (i.e., scaffolds) capable of supporting the required functional sites.
  • An algorithm e.g. ROSETTADESIGN
  • Computational ranking is performed to identify a relatively small number of potential enzyme designs, which can be empirically tested for the desired enzymatic efficiency. Potential candidates are then assayed experimentally. After experimental assay, designs can be further improved using in vitro evolution to identify more efficient variants.
  • the method is summarized diagrammatically in FIG. 1 .
  • a Diels-Alder active site model comprising a transition state model and/or substrate(s) and/or product model(s) is created computationally with proper positioning of catalytic functional groups.
  • the Diels-Alder reaction mechanism is a single step involving a cyclic redistribution of bonding electrons.
  • Frontier Orbital Theory shows that interaction of the highest occupied molecular orbital (HOMO) of the diene with the lowest unoccupied molecular orbital (LUMO) of the dienophile stabilizes the transition state and leads to charge transfer from diene to dienophile (30).
  • HOMO highest occupied molecular orbital
  • LUMO unoccupied molecular orbital
  • the reverse is also possible, and called inverse electron demand Diels-Alder reaction (as described elsewhere herein).
  • the interaction between the HOMO and LUMO, and the stability of the transition state relative to the ground state may be increased by a hydrogen bond acceptor interacting with the diene (to increase the HOMO energy and stabilize the positive charge accumulating in the transition state), and by a hydrogen bond donor interacting with an electron-withdrawing group of the dienophile (which lowers the LUMO energy and stabilizes the negative charge accumulating in the transition state), while the reverse is done for an inverse-electron demand Diels-Alder reaction.
  • a hydrophobic binding pocket that binds the core of the two substrates in the optimal relative orientation with appropriately placed hydrogen bond donors and acceptors, is believed to be an effective Diels-Alder catalyst.
  • the transition state model may be stabilized by substituent effects as described and/or by proximity effects, where substrates are bound in the proper position for catalysis.
  • the reactants or groups that make up the transition state generally will include a conjugated diene, and an alkene or alkyne, although the precise substrates may vary.
  • the diene may be a linear or cyclic diene. Where cyclic, the diene may be, for example, a five- or six-membered ring having two conjugated double bonds. The diene may have, for example, one or more heteroatoms, such N, O, and/or S. Generally, the diene must exist at least partially in the cis configuration, so as to support the Diels-Alder reaction.
  • the diene may be substituted with one or more substituents, the identity of which will depend on the desired product and desired catalytic mechanism.
  • Exemplary diene substituents have less than about 50, about 30, about 20, or about 15 atoms, and may contain one or more (e.g., 1-5) heteroatoms selected from O, N, or S, so long as such substituents do not interfere with the Diels-Alder reaction.
  • at least one substituent is capable of donating or accepting a hydrogen bond or has a polar group that can interact with the enzyme active site, thereby stabilizing the Diels-Alder transition state.
  • At least one substituent may be halogen (e.g., independently selected from Cl, Br, Fl, I), haloalkyl, alkyl (e.g., C 1-C5), —C(O)O, —C(O)OR, —COR, —CN, —C(R)N, —CH(O), —C(O)—, aryl (e.g., substituted or unsubstituted phenyl), OH, or —OR.
  • R may be any group, for example, containing about 20 or fewer atoms (e.g., 10 or fewer atoms).
  • R may represent a hydrogen or a carbon-containing substituent such as C1-5 alkyl (e.g., methyl), and may optionally include one or more (e.g., 1-3) heteroatoms independently selected from O, N, or S.
  • C1-5 alkyl e.g., methyl
  • heteroatoms independently selected from O, N, or S.
  • the diene has a substituent defined by —N(H)R, or a substituent that provides for a similar hydrogen-bonding geometry.
  • R may be as defined above, or as described elsewhere herein.
  • the dienophile may be substituted or unsubstituted, and the identity of such substituents will depend on the desired product and desired catalytic mechanism.
  • the dienophile in various embodiments is substituted with at least one electron-withdrawing group, making the dienophile more electrophilic.
  • the dienophile may be linear or cyclic, and generally includes an alkene or alkyne.
  • the dienophile may also have one or more heteroatoms selected from O, N, and/or S.
  • the substituent may be any group having less than about 30, about 20, or about 15 atoms, and may be suitable for withdrawing electron density from the carbon-carbon double or triple bond.
  • the electron-withdrawing group may contain one or more (e.g., 1-5) heteroatoms selected from O, N, or S.
  • the diene has at least one substituent selected from halogen (e.g., independently selected from Cl, Br, Fl, I), haloalkyl, —C(O)OR, —COR, —CN, —C(R)N, —CH(O), —C(O)—, aryl (e.g., substituted or unsubstituted phenyl), OH, and OR.
  • R may be any group, for example, containing about 30 or fewer, or 20 or fewer atoms (e.g., 10 or fewer).
  • R may represent a hydrogen or a carbon-containing substituent such as C1-5 alkyl (e.g., methyl), and may optionally include one or more (e.g., 1-3) heteroatoms independently selected from O, N, or S.
  • C1-5 alkyl e.g., methyl
  • heteroatoms independently selected from O, N, or S.
  • the dienophile has a substituent defined by —C(O)R, where R is H, linear or branched alkyl (e.g., C1-C5), or N(R) 2 .
  • R is H, linear or branched alkyl (e.g., C1-C5), or N(R) 2 .
  • the substituent provides for a similar hydrogen-bonding geometry as the C(O)R diene substituent described herein.
  • the dienophile is a substituted or unsubstituted C1-4 alkene, such as ethene or ethyne, 1,2 di-chloroethene or 2-buten-1,4-ol as shown in FIG. 21 .
  • transition state and placement of substrate(s) and product(s) in the active site model will be designed to account for the desired stereochemistry.
  • the transition state may be derived from the Diels-Alder reactants illustrated in FIG. 21 .
  • a transition state for an exemplary Diels-Alder reaction is shown in FIG. 4 , with asparagine or glutamine hydrogen bond acceptor to a diene substituent (NH carbamate), and a serine or tyrosine as a hydrogen bond donor to a carbonyl of the dienophile.
  • the diene and dienophile substituents shown may be replaced with any substituent described herein, where such substituents allow for stabilization of the transition state through interaction with amino acid functional groups (e.g., hydrogen bonding, electrostatic interaction, and van der waals and hydrophobic interactions).
  • a protein scaffold is then identified that could support the active site model (e.g., transition state, substrate(s), and/or product(s)) with desired catalytic functional groups.
  • the method employs a hashing algorithm, as described in Zanghellini et al., New Algorithms and an in silico Benchmark for Computational Enzyme Design, Protein Science 15:2785-2794 (2006); as well as WO 2009/076655, each of which is hereby incorporated by reference in its entirety.
  • first hashing method an inverse rotamer tree approach is used with a modified version of a known geometric hashing algorithm (Bachar et al. 1993), to find positions in a set of scaffolds that can support the catalytic site.
  • second hashing method based on iterative side chain placement and hashing in six-dimensional space, candidate catalytic sites in scaffolds are detected in linear time. Both methods are followed by the design of the pocket using the Rosetta design methodology.
  • an inverse rotamer tree is built up from the active site description, and the backbone coordinates of all the rotamer combinations are compared to backbone coordinates of the set of scaffolds using a geometric-hashing based algorithm.
  • side chain rotamers and the transition state (TS) model are sequentially placed at all scaffold positions, and the position of the TS model is recorded in a hash table. The hash table is then scanned for TS positions that are found when placing each of the catalytic side chains independently. These positions represent sites in the scaffolds where the specified active site can be successfully constructed.
  • the idea of the inverse rotamer tree is to convert the description of the active site in terms of functional groups into a description in terms of protein backbone coordinates, which can then be used to search a set of protein scaffolds, or to guide de novo scaffold design.
  • This technique is the inverse of the standard side chain packing problem, in which the positions of the backbone coordinates are known.
  • the algorithm employs a standard rotameric description of the side chains to solve the problem (Dunbrack and Cohen 1997); but, rather than building outward from the backbone coordinates, the side chains are grown backward from the functional group positions that are placed around the TS model in positions optimal for catalytic functionality.
  • This approach generates an inverse rotamer tree specifying the possible placements of the protein backbone around the TS model that are compatible with the specified active site, in the sense that the relevant amino acids can be placed to achieve the desired active site geometry.
  • each combination of backbone coordinates for the catalytic residues is searched against a set of scaffolds (a step subsequently referred to as matching) using a hashing-based approach.
  • a set of scaffolds for matching may include a plurality of scaffolds described in Table 1, or sequence and/or structural homologs thereof. In some embodiments, the set of scaffolds comprises at least 5, 10, 20, 100, or more of such scaffolds.
  • the first hashing algorithm begins by building a multiple key hash table.
  • the backbone coordinates e.g., N, Ca, C
  • the backbone coordinates for each pair of residues for each scaffold are mapped onto a unique key that is computed from the Ca-Cb distance and the [Ca, Cb] vector orientations.
  • all the scaffolds are mapped into a single hash in memory at the beginning of the program.
  • Each combination of backbone atom coordinates from the inverse rotamer tree is matched against the backbone distances and orientations stored in the hash table using a sub-graph isomorphism algorithm similar to that described by Russell (1998).
  • Matches are ranked based on their structural similarity (in RMSD) to the specified active site geometry and the absence of atomic clashes between the TS model, the placed catalytic side chains, and the protein backbone.
  • the idea of this approach is to build forward from the protein backbone to the TS model for each catalytic side chain independently, and then to identify TS placements compatible with placement of each catalytic residue.
  • the method includes ligand orientation, as well as center of mass coordinates. The following first describes the storage of the position of the TS model for each catalytic side chain rotamer placed at each position using a hash table and next, the processing of the hash table to extract sets of positions compatible with the specified active site geometry. Finally, performance enhancements to the method using pre-computed grids to restrict TS placement to clefts and pockets in the scaffolds, and to speed up the evaluation of atomic clashes with the protein backbone are discussed.
  • a set of potential active site positions is predefined, either all positions in the protein, or positions lining cavities or small molecule binding sites.
  • all rotamers from the Dunbrack backbone dependent library are placed at each position. If there is no clash with the protein backbone, the TS model for the reaction is positioned as specified in the catalytic site definition.
  • a large set of TS model placements are considered; the fineness of the sampling around the varying degrees of freedom is illustrated in FIG. 5 .
  • Empirical testing is performed to build and test the actual enzymatic activity of selected sequences. If desired, those sequences showing promise are further manipulated using the technique of in vitro evolution, and the results are then empirically tested.
  • the TS model and the catalytic side chains are refined to eliminate clashes and optimize the catalytic geometry. Then, the identity and conformations of amino acid residues located near the active site are optimized using a software module referred to as ROSETTADESIGN. Finally, the designs are ranked based on the computed TS binding energy, considering only designs where the catalytic constraints are satisfied. However, it should be emphasized that determination of the catalytic efficacy of a design requires experimental characterization.
  • the center of mass of the TS model may be restricted to clefts or pockets that are likely to be large enough to comprise a viable active site.
  • a square grid box is first constructed that covers the regions targeted for active site design. This grid is then trimmed to remove all the grid points that are ⁇ 2.25 ⁇ from any protein backbone atom. Any residue on the protein backbone that has a Ca-Cb vector pointing toward one of those grid points and a Ca ⁇ 3.5 ⁇ from any grid point is then included in the set of active site positions.
  • the use of the grid does not substantially reduce the number of matches found, but it considerably speeds up the search process by eliminating regions unlikely to contribute high ranking active site designs.
  • a “backbone” grid is constructed that contains points that are ⁇ 2.25 ⁇ from any backbone atom. TS model placements for which atoms overlap the backbone grid are not included in the hash.
  • residues around the TS model are truncated to glycines or alanines.
  • modification of truncation to alanine (Ala) may result in enzymes that are easier to handle.
  • the initial placements of the TS model and catalytic side chain conformations are optimized by rigid body minimization followed by side chain minimization using ROSETTA (Gray et al. 2003; Wang et al. 2005).
  • the minimization step leads to pockets in which a non-clashing TS model is placed with catalytic side chains positioned with functional atoms close to the optimal geometry required for catalysis. It is then necessary to design the surrounding, non-catalytic protein residues to maximally stabilize the transition state.
  • the conformations and identities of residues surrounding the TS model are optimized using Monte Carlo simulated annealing as described previously (Kuhlman and Baker 2000).
  • the invention involves making Diels-Alder enzymes by mutation of the scaffolds and enzymes described herein (e.g., SEQ ID NOS: 2, 4, 6, 8, and 9), or sequence or structural homologs thereof, to accommodate desired Diels-Alder substrates. Positions of such mutations and their likely involvement in substrate, transition state, or product binding, or catalytic activity, have been described.
  • the method comprises introducing, in the amino acid sequence of SEQ ID NO:4 (DA — 20 — 00), or a homolog thereof as described herein, from 1 to about 30, or about 1 to 20, or about 1 to 10 amino acid substitutions, insertions, and/or deletions (collectively).
  • Such mutations are made to establish or enhance catalytic efficiency for a particular Diels-Alder substrate, such as those described herein.
  • the Diels-Alder catalyst in accordance with these embodiments contains the catalytic residues at positions corresponding to positions 121 and 195 of SEQ ID NO:4, and otherwise has been modified in the substrate binding pocket, to bind the Diels-Alder substrates of choice.
  • the method comprises introducing, in the amino acid sequence of SEQ ID NO:6 (DA — 20 — 04), or a homolog thereof as described herein, from 1 to about 30, or about 1 to 20, or about 1 to 10 amino acid substitutions, insertions, and/or deletions (collectively).
  • Such mutations are made to establish or enhance catalytic efficiency for a particular Diels-Alder substrate, such as those described herein.
  • the Diels-Alder catalyst in accordance with these embodiments contains the catalytic residues at positions corresponding to positions 121 and 195 of SEQ ID NO:6, and otherwise has been modified in the substrate binding pocket, to bind the Diels-Alder substrates of choice.
  • the method comprises introducing, in the amino acid sequence of SEQ ID NO:8 (DA — 20 — 10), or a homolog thereof as described herein, from 1 to about 30, or about 1 to 20, or about 1 to 10 amino acid substitutions, insertions, and/or deletions (collectively).
  • Such mutations are made to establish or enhance catalytic efficiency for a particular Diels-Alder substrate, such as those described herein.
  • the Diels-Alder catalyst in accordance with these embodiments contains the catalytic residues at positions corresponding to positions 121 and 195 of SEQ ID NO:8, and otherwise has been modified in the substrate binding pocket, to bind the Diels-Alder substrates of choice.
  • the method comprises introducing, in the amino acid sequence of SEQ ID NO:9, or a homolog thereof as described herein, from 1 to about 30, or about 1 to 20, or about 1 to 10 amino acid substitutions, insertions, and/or deletions (collectively). Such mutations are made to establish or enhance catalytic efficiency for a particular Diels-Alder substrate, such as those described herein.
  • the Diels-Alder catalyst may be designed to stabilize the transition state for a variety of Diels-Alder reactants. That is, amino acid substitutions, insertions and/or deletions with respect to the scaffold, or with respect to the Diels-Alder catalyzing enzymes of SEQ ID NOS: 4, 6, 8, and 9 may be selected with regard to a particular Diels-Alder reaction.
  • the Diels-Alder catalysts made or manufactured in accordance with these aspects are expressed and purified and/or isolated using know techniques, including in some embodiments on an industrial scale.
  • the enzymes may be expressed in any bacterial or eukaryotic expression system, including E. coli or yeast expression systems, and may be purified using any suitable chromatographic step or steps, including affinity, size and/or charge-based chromatography.
  • the Rosetta computational design methodology has been used to design novel enzymes (1,4) that catalyze bond breaking reactions, but bimolecular bond forming reactions present a new challenge as both substrates must be bound in the proper relative orientations.
  • previous successes with computational enzyme design have involved general acid-base catalysis and covalent catalysis, but the Diels-Alder reaction instead can be primarily influenced by modulation of molecular orbital energies (30).
  • These examples show the design and structure of intermolecular Diels-Alder enzyme catalysts, capable of catalyzing, for example, the well-studied model Diels-Alder reaction between 4-carboxybenzyl trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide ( FIG. 2 , substrates 1 and 2, respectively) (32,10).
  • the general protocol for Rosetta enzyme design is comprised of four steps. First, an ensemble of transition states for the reaction is built, with optimal placement of the catalytic functional groups. Each transition structure is then matched against a library of protein scaffolds using the ROSETTAMATCH algorithm. The resulting matches are then redesigned using ROSETTADESIGN so as to maximally stabilize the transition state and the placement of the catalytic residues. Designs are then ranked based on the binding energy and the satisfaction of the catalytic geometry, clustered and visually inspected. When needed, the resulting designs are then modified in silico through point mutants to improve shape complementarity (e.g., hydrophobic burial vs. more polar interfaces).
  • the basic computational design methodology has been described in Zanghellini et al., New algorithms and an in silico Benchmark for computational enzyme design, Protein Science 15:2785-2794 (2006), which is hereby incorporated by reference in its entirety.
  • the first step in de novo enzyme design is to decide on a catalytic mechanism and an associated ideal active site.
  • Frontier Orbital Theory shows that the interaction of the highest occupied molecular orbital (HOMO) of the diene with the lowest unoccupied molecular orbital (LUMO) of the dienophile stabilizes the transition state and leads to charge transfer from diene to dienophile (30).
  • the Rosetta enzyme design methodology was employed. This approach requires three-dimensional atomic models of minimal active sites consisting of the reaction transition state and protein functional groups carrying out catalysis.
  • a carbonyl oxygen from a glutamine or asparagine to hydrogen bond with the N—H of the diene carbamate
  • a hydroxyl from a serine, threonine, or tyrosine to hydrogen bond with the carbonyl oxygen of the dienophile amide moiety
  • the transition state coordinates were obtained using quantum chemistry (QM) calculations in the presence of the specified hydrogen bond donors and acceptors. The calculations were carried out with an acetate molecule to represent the carboxylic group of an aspartate or glutamate amino acid hydrogen-bonding the N—H of the diene carbamate.
  • QM quantum chemistry
  • the acid group acting as an electron donor to the diene would be difficult to desolvate, making binding of the diene difficult. Therefore, the acid group hydrogen bonding to the N—H carbamate of the diene was replaced by an amide group, representing the functional group of a glutamine or asparagine amino acid. This change was predicted to decrease the cost of desolvation, and improve binding to the diene.
  • the oxyanion hole was replaced by a single hydroxyl group representing a serine, threonine, or tyrosine amino acid. The hydroxyl was placed in a standard hydrogen bonding geometry from the carbonyl group.
  • the program SPARTAN was used to replace the methyl group on the diene with a caboxybenzyl group, since 4-Carboxybenzyl trans-1,3-butadiene-1-carbamate is the actual substrate used in the experimental assay.
  • the diene, 4-Carboxybenzyl trans-1,3-butadiene-1-carbamate can adopt different distinct conformers corresponding to different values of the dihedrals ⁇ 1 and ⁇ 2 ( FIG. 3 ). Since continuous optimization of these free degrees of freedom during the active site placement search is impossible, a discrete ensemble of conformers was generated where each of the dihedral is uniformly sampled every 60 degrees. Each conformation generated was checked for an internal clash, defined as a distance of less than 3.2 ⁇ between two atoms separated by more than two bonds. This clash filter reduced the number of transition states from a total of 36 down to a total of 15 final transition structures.
  • the geometry of the transition state structure in the carbon-carbon bond forming region differs significantly from the geometries of the product or the two substrates placed in the productive orientation. Although it is desired to optimally stabilize the transition state to increase catalytic rate, it is also necessary for the protein pocket to be able to accommodate (1) the two substrates bound in the relative orientation that will lead to the transition state and (2) the product of the reaction. To ensure that the designed model would accommodate all these steps along the reaction pathway, composite active site descriptions were built using the program SPARTAN. A model of the two substrates optimally oriented for catalysis, and the product of the reaction was generated.
  • the chair conformation is favored at equilibrium in solution, but the two carbon-carbon bonds formed by the Diels-Alder reaction leads initially to a boat conformation that then relaxes to the chair conformation. Therefore, the product model included in the composite transition state is in the boat conformation.
  • the substrate and product models were then overlaid with the quantum mechanical derived transition state model in order to generate a single composite active site model.
  • the choice of the superimposition method was somewhat arbitrary.
  • the three models (substrate/transition state/product) are superimposed on carbamate portion of the molecule since it is part of the core of the reaction and precise interactions were to be made with that portion of the molecule.
  • a diverse ensemble of 1.3 ⁇ 10 11 distinct minimal active sites were generated by systematically varying the identity and rotameric state of the catalytic side chains, the hydrogen bonding geometry between these residues and the transition state, and the internal degrees of freedom of the transition state.
  • a library of 207 protein scaffolds was used for active site placement (see Table 1).
  • the scaffolds were selected such that a high-resolution crystal structure was available, and that expression in Escherichia coli was possible and documented.
  • Each scaffold is equipped with one grid to mark the spatial extension of the pocket, and one grid that discretizes the pocket backbone positions for speeding up Van der Waals clash calculations (1).
  • the ROSETTAMATCH algorithm searches the library of protein scaffolds, stripped of all amino acid side chains, for pockets that fit the ligand in the correct orientation relative to each of the catalytic residues when coming off specified positions of the scaffold backbone.
  • ROSETTAMATCH algorithm employs hashing to identify positions in a set of input protein scaffolds that support the construction of a specified constellation of catalytic residues. For each composite active site description, candidate catalytic sites were generated in the scaffold library. At each position in the active site pockets on each scaffold, each rotamer for each catalytic sidechain is placed and an ensemble of composite transition structures are placed so as to satisfy the geometrical parameters as described for the active site. The positions of the composite transition structure within the scaffold backbone are recorded in a six-dimensional (6D) hash.
  • 6D six-dimensional
  • an ensemble of rigid-body placements of the transition state structure is generated from the functional atoms of the sidechain, based on the parameters summarized in FIGS. 5 and 6 .
  • thousands of different rigid-body orientations of the transition state structure are placed (around 7700 for the first catalytic residue, and around 2600 for the second).
  • the total number of combinations evaluated by the algorithm at each position in the scaffold is more than 75,000,000 combinations for the first catalytic residue, and more than 200,000 for the second.
  • the active site placement problem is one of combinatorial explosion, with a theoretical search space of more than 1.3 ⁇ 10 11 possibilities for the 15 transition state structures.
  • the algorithm efficiently decreases the complexity of the search by aggressive pruning.
  • the ensembles of rigid-body placement are first further trimmed based on their steric clash with the backbone (N,C,C ⁇ ,O) and C ⁇ atoms. Previous uses of ROSETTAMATCH did not consider clash checks with the C ⁇ , but only the protein backbone heavy atoms.
  • the hash table which scales linearly with the number of scaffold positions and number of sidechain rotamers, the hash is examined for transition structure positions compatible with all catalytic constraints; such positions are termed “matches”.
  • the size of the bins for hashing are set to 0.6 ⁇ (hashing on the center of mass rigid-body) and 10°.
  • ROSETTAMATCH checks that the placed catalytic side-chains do not have significant steric repulsion with each other. Every match with a total steric repulsion greater than 1.0 energy unit was discarded. To evaluate whether a match was sufficiently buried, key atoms of the transition state structure (the atoms corresponding, in the transition state, to the product cyclohexene carbons) are tested for being in the grid that defines the active site pocket of each scaffold. To ensure even sampling, and avoid collecting millions of matches at the same positions, only the first 250 matches that occur in the same scaffold with the same catalytic residues at the same position in the pocket are kept.
  • the final number of matches generated by the algorithm was on the order of 500,000. Compared with the theoretical number of active sites sampled (1.3 ⁇ 10 11 ), this result shows that the likelihood of finding the specific geometrical arrangement of a minimal active site in around 200 scaffolds is on the order of 3 per million.
  • the Diels-Alder reactions were performed in PBS, at 25° C., with 4% DMSO.
  • the DMSO came from the diene, for which the stock solution (100 mM) was made in DMSO and always diluted such that the final reaction would contain 4% DMSO.
  • time points were taken of the reaction (5 ⁇ L) and quenched in a solution of 80:20 acetonitrile:water with 0.1% formic acid and 1 mM Benzoic Acid (95 ⁇ L).
  • DA — 20 — 00 One design (DA — 20 — 00) showed an increased activity over the background reaction.
  • This active design (DA — 20 — 00) was created from a 6-bladed beta barrel propeller scaffold (PDB-ID 1E1A; a diisopropylfluorophosphatase from Loligo vulgaris ).
  • the amino acid sequence of the scaffold with respect to the original design DA — 20 — 00 is shown in FIG. 7 .
  • the tertiary structure of the scaffold is illustrated in FIG. 8 , and local secondary structures displayed in FIG. 9 .
  • the rest of the pocket is lined with hydrophobic residues that form a tight shape-complementary surface ( FIG. 10A ).
  • a second enzyme based on a ketosteroid isomerase scaffold, was also identified.
  • the amino acid sequence of this enzyme is shown in FIG. 20 , with active site residues identified.
  • site-directed mutagenesis was performed in order to optimize the active site. Active site mutations were picked through a visual assessment of the designed active site, in which positions that had the potential to make new contacts to the diene or dienophile substrate upon mutation were identified. All mutagenesis was performed following the Kunkel protocol (8), using oligonucleotides designed using the Stratagene primer design online tool. After sequence verification of the mutated genes, protein was expressed and purified as described above.
  • the original active Diels-Alder enzyme (DA — 20 — 00) and an inactive variant of the same protein (DA — 20 — 01) were expressed and assayed, in triplicate, along side as a control.
  • a second round of active site optimization was conducted on DA — 20 — 04.
  • the number of mutations screened was increased to 176 variants in order to allow for a larger breath of mutations to be made. Mutations were picked as in the first round of mutagenesis, except that we also had MD simulations to for guidance. Most noteworthy is the variation of residue 272. Additional mutations were allowed at this position since molecular dynamic simulations suggested that a larger amino acid here would bolster the catalytic tyrosine (Y121) into place.
  • This culture was grown for 2-3 hours at 37° C., after which 50 ⁇ L of 10 mM IPTG was added to each well (0.5 mM Final) in order to induce expression.
  • the cells were then grown for 24 hours at 18° C., harvested, and stored at ⁇ 20° C. until ready for assaying.
  • crude cell lysate was prepared by resuspending the cells in 500 uL of PBS and lysing through 3 freeze/thaw cycles (15 minutes at ⁇ 80° C., 40 minutes at 25° C.), followed by centrifugation at 4000 rpm for 30 minutes. 434 of the supernatant was incubated with 1 mM diene and 10 mM dienophile for two hours at room temperature. Samples from each reaction were then quenched and product detected using a liquid chromatography-tandem mass spectroscopy.
  • DA — 20 — 10 showed the most significant activity and was therefore used as the new WT.
  • the variant and the combinatorial variants that make up DA — 20 — 10 were sequenced, expressed, purified, and assayed using the standard protocol described for the first round of mutagenesis. The activity observed for each of the validated mutants is shown in FIG. 13 .
  • the triple mutant DA — 20 — 10 produced 154.9 nM of product/hour under the test conditions, as compared to 21.9 for DA — 20 — 04.
  • An alignment of DA — 20 — 04 and DA — 20 — 10 is shown in FIG. 14 .
  • residues contacting the transition state in DA — 20 — 00 were mutated individually to sets of residues predicted to retain or improve transition state binding and bolster the two catalytic residues.
  • Six mutations were found that in combination increase overall catalytic efficiency by roughly 2200-fold over the original design model, DA — 20 — 00; the protein with these six additional mutations is designated DA — 20 — 10 ( FIGS. 12 and 14 ).
  • Three of the mutations ( FIG. 6C ) likely improve the packing around the transition state (A741, A21 T) or catalytic glutamine (A173C).
  • the kinetics of the DA — 20 — 10 catalyzed reaction was characterized by measuring the dependence of the reaction velocity on the concentration of both diene and dienophile. See FIG. 15 .
  • the kinetic parameters for DA — 20 — 10 and DA — 20 — 00, including with respect to various catalytic antibodies, are summarized in Table 5.
  • the effective molarity (kcat/kuncat) of DA — 20 — 10 is 100 M
  • the rate enhancement (kcat/(kuncat*KM_diene)) in the presence of saturating dienophile and limiting diene is 1.0 ⁇ 10 5 .
  • DA — 20 — 10 proceeds for more than 30 turnovers before the enzyme loses activity.
  • At higher enzyme concentrations more than 80% of the diene substrate is converted to product.
  • the errors represent the calculated 95% confidence interval.
  • the parameter a describes the cooperativity of binding between the two substrates: a value greater than one indicates negative cooperativity, whereas a value lower then one indicates positive cooperativity. ⁇ could not be determined for DA — 20 — 00.
  • Kinetic parameters for catalytic antibodies 7D4 and 4D5 were taken from (10, 17). The k uncat for the Diels-Alder reaction at 298K was found to be 2.44 ⁇ 10 2 M ⁇ 1 hr ⁇ 1 , in good agreement with the previously reported value at 310K of 4.29 ⁇ 10 ⁇ 2 M ⁇ 1 hr ⁇ 1 .
  • the kinetic parameters of DA — 20 — 10 compare favorably with the best catalytic antibodies elicited for this Diels-Alder reaction (Table 4).
  • the kcat of DA — 20 — 10 is 10-fold greater than those of 7D4 (10) and 4D5 (17), but this may be an underestimate because the catalytic antibodies were measured at 310K rather than 298K.
  • the KM for the diene is in the same millimolar range as the catalytic antibodies, but the KM for the dienophile is one order of magnitude worse, which is not surprising given the power of the immune system to produce high affinity binding proteins to arbitrary ligands.
  • the rate enhancement with saturating diene and limiting dienophile (kcat/(kuncat*KM_dienophile), 1.7 ⁇ 10 3 ) is similar to those of the antibodies, but the effective molarity (kcat/kuncat) and the rate enhancement with saturating dienophile and limiting diene (kcat/(kuncat*KM_diene)) are both 20-fold greater than for either antibody.
  • each dienophile from FIG. 19 was either purchased or synthesized.
  • the dienophiles were assayed in the presence or absence of 60 ⁇ M DA — 20 — 10 or the H287N mutant over 30 hours with 10 mM dienophile and 0.2 mM diene at 298K in a PBS solution.
  • a sample of the reaction was taken at four different time points, quenched, and filtered as previously described.
  • To detect product a liquid chromatography-mass spectroscopy assay was used.
  • the mass spectrometer was used in a selective-ion-monitoring mode and set to specifically detect the expected product from each reaction.
  • the average area of each detected peak formed per hour was used to determine the relative amount of product formed with and without enzyme.
  • the relative peak areas formed per hour are shown in FIG. 19 .
  • the Diels-Alder reaction studied here can, in principle, produce eight different isomeric products, four of which are experimentally observed in the reaction in solution FIG. 19 (10).
  • the computational design was directed at the transition state that yields the endo-Re product (3R,4S), which comprises 47% percent of the total product formed in the uncatalyzed reaction. Consistent with the design, DA — 20 — 10 only catalyzes the formation of the expected 3R,4S product (>97%).
  • the high diastereo- and enantioselectivity of DA — 20 — 10 demonstrates the ability of the computational enzyme design protocol disclosed herein to control reaction stereoselectivity.
  • This Diels-Alderase is the first computationally designed enzyme that catalyzes a synthetically important abiological reaction, the formation of two carbon-carbon bonds from two distinct molecules, and exhibits both strong stereoselectivity and substrate specificity.
  • the agreement between the designed and experimentally observed substrate specificity and stereoselectivity of the enzyme is notable given the importance of selectivity in organic reactions.
  • the capability to rationally control both substrate specificity and stereoselectivity via designed enzymes opens up new avenues of research in both basic and applied chemistry.
  • biocatalysis for drug manufacturing is extremely attractive to the chemical and pharmaceutical industries, its use has been limited by the ability to discover or evolve enzymes with the desired substrate specificity and stereoselectivity. Our results suggest that computational enzyme design provides a solution to this problem.
  • Redesign of either DA — 20 — 00, DA — 20 — 04 or DA — 20 — 10 for new substrates involves the following steps.
  • This active site may contain new active site residues (i.e. amino-acid to originate from the protein) or not, depending on the substituent on the diene and dienophile of interest.
  • this active site can be placed in the scaffold of DA — 20 — 00, DA — 20 — 04 or DA — 20 — 10 by placing the active site into the protein pocket using a hashing-based active site placement method as disclosed herein, followed by design and minimization of the rest of the pocket.
  • the active site may be placed in the scaffold by superimposing the “catalytic” core of the active site model for the new substrate(s)/transition state/product(s) to the catalytic core of the active site previously placed in DA — 20 — 00, DA — 20 — 04 and DA — 20 — 10.
  • the catalytic core may be comprised of the two double bonds of the diene, the double bond of the dienophile, the two carbon-carbon bond being formed as the reaction proceeds.
  • Other superimposition techniques are possible, for instance superimposition on substituents common to the new substrate(s)/transition state/product(s) and the one that were used for the original design of DA — 20 — 00, DA — 20 — 04 and DA — 20 — 10.
  • the active site may be placed by altering the backbone of the scaffolds of DA — 20 — 00, DA — 20 — 04, and DA — 20 — 10 to accommodate the new substrate(s)/transition state/product(s).
  • Alteration of the backbone may require the generation of a variable number of backbone models, typically on the order of 1,000 to 1,000,000.
  • the minimization step may include minimizing the substrate(s)/transition state/product(s) internal degrees of freedom, minimizing the protein side-chain and backbone degrees of freedom using standard force-fields, as well as rigid-body placement of the substrate(s)/transition state/product(s).
  • the minimization step is followed by design of the rest of the protein pocket.
  • the design step may include any number of variable positions, and an additional minimization step in the context of new sequences optimized by the design step. This subsequent minimization step may include the same features as above.
  • Promising designs are then ranked and selected for testing.
  • Reaction (1) produces an intermediate used for the production of TAMIFLU.
  • Reaction (2) leads to the production of a building block useful for the manufacture of the polymer PET.
  • Reaction (3) can be used for the manufacture of vitamin B6.
  • Reaction (4) is a reaction commonly referred to as “click chemistry.”
  • the enzymes produced in Example 1 may be optimized to catalyze such Diels-Alder reactions, using the information and methods disclosed herein.
  • the active site of the enzyme designs disclosed herein may be altered to be less hydrophobic to accommodate more polar structures and/or to provide additional contacts for the Diels-Alder substrates and/or transition states, and may include additional hydrogen bonding and electrostatic contacts.
  • the active site of the designs can be altered to present a more hydrophobic interface to one or both of the substrates, such as ethylene.

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